1. Field of the Invention
The present invention relates to functional polypeptide compositions originating from silk protein excellent for promoting cell growth, extensibility and the like, a production method thereof and use thereof in the fields of medicament, quasi drug, cosmetics as a material for skin care, food and the like.
2. Description of the related Art
Since silk threads have been used as a surgical suture from the old days, silk protein is regarded as a biocompatible material, and developments have recently become active for new uses of silk threads in various fields other than that of clothing.
For example, as available fields, silk threads are solubilized to form an aqueous silk protein solution, followed by conversion to powder by coagulation, drying, grinding, etc., for additives of cosmetics; the aqueous silk protein solution is made into a film-like material by casting on a plate and the like for cell culture bed, wound-covering and coating material; and the aqueous silk protein solution is made into a gel-like material for use in food and cosmetics. The developments of these uses are pursued (see Patent literatures 1 to 11).
As described above, silk materials are processed and used in the forms of powder, film, gel and the like after silk threads are solubilized to form an aqueous silk protein solution.
In the processes of these silk materials, neutral salt is mainly used for solubilizing silk threads. However, there is no description that the aqueous solution of neutral salt of silk protein has a property of human cell growth promotion.
Further, during the development of new silk materials, it has come to be said that silk protein has a variety of functions such as cell growth, antioxidation, germicidal action, alcohol digestion and anticoagulation of blood.
The present inventors paid attention to the cell growth function associated with silk protein and have pursued the development and study of understanding of its function with the aim of utilizing cocoon filaments or silk threads as a skin care material for wound-covering materials and cosmetics after these are solubilized and then converted into powder, film, gel and the like (see Patent literatures 12 to 14).
Furthermore, the present inventors had separated, purified and recovered the components constituting silk protein and evaluated their functions, studying which site or structure in silk protein was responsible for the cell growth function. As the result, it was found that the fibroin H-chain (see Patent literature 15) and L-chain (see Patent literature 16) and the a component of sericin (see Patent literature 17), both constituting silk protein, have a growth-promoting action on fibroblasts originating from normal human skin.
They are particularly excellent for cell adhesion in the cell growth process.
Fibroin in which the H- and L-chains are held together via SS bonds having a molecular weight of 370,000 daltons and the sericin a having a molecular weight of 400,000 daltons are called undegraded silk protein.
On the other hand, silk threads are used for fiber, besides the field of clothing, in the fields of medical care (surgical suture for surgery), cosmetics (puff) and the like.
It has recently been recognized that the molecular weight of silk protein decreases during these processes of cocoon and raw silk.
Moreover, in the processes of converting cocoon filaments or silk threads into powder, film, gel and the like (particularly, the solubilizing process of cocoon filaments or silk threads), the molecular weight of silk proteins was found to decrease (see Patent literatures 18 and 19).
An electrophoretogram of silk protein with molecular weight decreased by conventional processes of silk shows only a broad band with a smear in a molecular weight range between 10,000 and 200,000 daltons.
Degraded products of silk protein having lower than ca. 5,000 daltons are eliminated during the processes of dialysis and the like. Therefore, it is considered that the molecular weight of silk protein has been decreased up to amino acid and peptide levels during the processes. Here, polypeptide means having amino acid residues not less than 30 and peptide means having amino acid residues less than 30.
In addition, it has been found that silk proteins with reduced molecular weights in such ways are reduced in growth-promoting activity for human cells (see Patent literature 14). As to the cell growth rates of silk fibroin and sericin, they show highest values in their undegraded states. When an average molecular weight is decreased to about 200,000 daltons, the cell growth rate becomes about half of that at the time when they are in undegraded states, and when the molecular weight is decreased to 20,000 to 40,000 daltons, the cell growth-promoting activity is hardly present.
This reason may be that substances inhibiting cell growth are generated as their molecular weights are reduced.
As to the reasons for the inhibition of cell growth by silk protein, it is considered that peptide bonds of protein are generally cleaved in a complex fashion and heterogeneously by acid, alkali, light (ultraviolet light, radiation and the like), heat or the like, thereby reducing its molecular weight, or that modification of the side chains of amino acid residues (oxidization, halogenation, deamidation or the like) takes place, or the like.
In other words, treatment with acid, alkali, light, heat or the like during the steps of silk processing causes reduction in the molecular weights due to cleavage of nonspecific peptide bonds as well as increased inhibition of cell growth in spite of excellence for cell growth promotion of undegraded fibroin and sericin.
Accordingly, in order to use the function of cell growth promotion of silk protein, it is preferred to use undegraded silk fibroin, sericin, the H-chain (about 350,000 daltons) and L-chain of fibroin, sericin a (about 400,000 daltons), or the like.
However, when the physical properties of silk protein are taken into consideration, fibroin and sericin in solution have properties that the higher the molecular weights of fibroin and sericin are, the more easily they become fibers (crystallization) by shear at the time of vibration or stirring.
Substances transformed to fibers become water insoluble aggregates.
In particular, undegraded silk protein (fibroin and/or sericin) tends to form gel (soft crystal containing water) in solution even if there is no shear.
An aqueous solution of silk protein having a molecular weight not lower than 200,000 daltons dissolved with a neutral salt becomes gel during desalting.
The gel gradually hardens, and it becomes harder when it is stirred.
Fine fiber formation easily takes place by a slight shear as described above, which causes to lower availability (touch, extensibility) of ointment and cosmetics (cream, emulsion, conditioner, and the like), resulting in poor applicability to skin care materials.
For example, when an aqueous solution of undegraded fibroin or an aqueous solution of fibroin having a molecular weight larger than ca. 300,000 daltons is lightly rubbed on hands, water insoluble aggregates of about 0.5 to 2 mm are easily formed. Even if it has a molecular weight of about 200,000 daltons or larger, aggregates are formed when it is rubbed hard, presenting lack of extensibility.
On the other hand, as the molecular weight of silk protein decreases, fiber formation by friction and the like may not occur.
In particular, when a molecular weight of silk protein becomes lower than 200,000 daltons, particularly from 50,000 to 100,000 daltons, no fiber aggregates in a ball shape are not formed even if the aqueous silk protein solution is rubbed hard. For this reason, the silk protein can become an excellent material in physical properties as a material for cosmetics or the like.
However, in silk threads for silk fabric and the like obtained by conventional cocoon processing, the H-chain of fibroin cannot be confirmed and the L-chain thereof is degraded to a level hardly confirmed.
The cell growth-promoting activity of such fibroin is about half of or less than half of undegraded fibroin.
The function of cell growth of silk protein is decreased to less than or equal to one-half of that of undegraded silk protein. However, fibroin and sericin have been used as their molecular weights were reduced during the silk manufacturing processes or the like.
In other words, the conventional uses of silk fibroin and sericin were aimed at their easy use and availability rather than their cell growth properties, and fibroin and sericin having molecular weights lower than 200,000, mainly less than 100,000, have been used.    [Patent Literature 1]    Kokoku (Jpn. examined patent publication) No. 40-24920    [Patent Literature 2]    Kokai (Jpn. unexamined patent publication) No. 62-415    [Patent Literature 3]    Kokoku (Jpn. examined patent publication) No. 1-44320    [Patent Literature 4]    Kokai (Jpn. unexamined patent publication) No. 1-254164    [Patent Literature 5]    Kokai (Jpn. unexamined patent publication) No. 1-256351    [Patent Literature 6]    Kokoku (Jpn. examined patent publication) No. 4-202435    [Patent Literature 7]    Kokoku (Jpn. examined patent publication) No. 5-83292    [Patent Literature 8]    Kokoku (Jpn. examined patent publication) No. 6-4679    [Patent Literature 9]    Kokai (Jpn. unexamined patent publication) No. 8-143595    [Patent Literature 10]    Kokai (Jpn. unexamined patent publication) No. 11-139986    [Patent Literature 11]    Kokai (Jpn. unexamined patent publication) No. 11-276876    [Patent Literature 12]    U.S. Pat. No. 2,997,758    [Patent Literature 13]    U.S. Pat. No. 2,990,239    [Patent Literature 14]    Patent application No. 2002-230656    [Patent Literature 15]    Kokai (Jpn. unexamined patent publication) No. 2001-163899    [Patent Literature 16]    patent application No. 2001-180169 (WO02/102845A1 WO publication)    [Patent Literature 17]    Kokai (Jpn. unexamined patent publication) No. 2002-128691    [Nonpatent Literature 1]    By H. Yamada et al.: Materials Science & Engineering C, 14, P. 41–46 (2001)    [Nonpatent Literature 2]    K. Tsubouchi, H. Yamada, Y. Takasu: The Japanese Society of Sericulture Science academic journal Vol. 71, No. 1, P. 1–5 (2002)